Down syndrome (DS) is caused by trisomy for human chromosome 21 (HSA21). The Down syndrome critical region hypothesis suggests that most DS features result from one or a few dosage sensitive genes located in critical regions on the chromosome. We performed the first direct test of this hypothesis by creating mice with a duplication or deletion of a critical region associated with a variety of DS facial features. Our analysis shows that genes from the critical region are not sufficient and are largely unnecessary to produce these complex features in mice, thus refuting the DSCR hypothesis. Molecular level studies remain important for DS, but explanations of the etiology of DS phenotypes must include the critical dimensions of where and when genes are expressed in development, and a realization that trisomy-induced events in one cells may initiate a cascade of changes in other cells, independent of additional gene dosage effects. Animal models are essential to these further studies, but existing mouse models for DS are inadequate. They do not represent the complete genetic insult and range of phenotypes in DS. Further, they are difficult to use, discouraging consideration of DS by the wider scientific community, something that is critical in dealing with a problem as complicated as trisomy 21. We will use chromosome engineering techniques optimized in the previous award period to build three new mouse models for DS that together contain three copies of all mouse orthologs of HSA21 genes. We will characterize them for additional, quantifiable features with parallels to DS. These more complete genetic models will simplify both husbandry and typing of trisomic mice, making them accessible to a much larger community, and will greatly facilitate developmental studies that are an essential to understanding and finally ameliorating the effects of trisomy. In the last award period, we made a second fundamental discovery by showing that the trisomic transcriptome is characterized by small changes in a large percentage of disomic genes. These changes are for the most part not individually significant, but collectively, they robustly distinguish trisomic and euploid transcriptomes. This finding that hundreds of disomic genes are expressed differently in trisomic vs. euploid cells has been confirmed in a purified population of cultured granule cell precursors. In addition, we provided direct evidence to prove the observation from epidemiological studies that individuals with trisomy 21 have a significantly lower than expected incidence of solid tumors, and narrowed from 350 to 33 the number of candidate resistance genes that are sufficient to provide protection. We will use new models to determine the genetic basis for this resistance.